Acanthamoeba, like many free-living protozoa, contains a osmoregulatory organelle called the contractile vacuole. This intracellular specialized vacuole accumulates water and expels it from the organism. The mechanism of water accumulation by the contractile vacuole is totally unknown. In collaboration with Richard Leapman, NIH, NCRR, BEIP, we are attempting to determine the ionic content of the vacuole in order to better understand the mechanism of water secretion. The methods employed include rapid freezing, cryomicrotomy and analytical electron microscopy. Analysis for common cellular elements (Na,K,Mg,P,S,Cl) was carried out on rapidly frozen, cryosectioned material that was dried from the frozen state in the vacuum of the electron microscope. Mitochondria and cytoplasm of Acanthamoeba have an ion composition similar to other cell types. In digestive vacuoles Na and K are present in close to eqimolar ratios and the major counter ion is Cl. Because the contractile vacuole contains only water and salts (i.e. no organic material), the salts aggregate on freeze drying and the results so far give only the ion ratios. The major ion is Na and the Na:K is about 6:1. Accurate knowlege of the ion content of the contractile vacuole is important for designing experiments to elucidate the mechanism of contractile vacuole function.